Cell-specific targeting for delivery of effector moieties such as diagnostic or therapeutic agents is a widely researched field and has led to the development of non-invasive diagnostic and/or therapeutic medical applications. In particular in the field of nuclear medicine procedures and treatments, which employ radioactive materials emitting electromagnetic radiations as γ rays or particle emitting radiation, selective localization of these radioactive materials in targeted cells or tissues is required to achieve either high signal intensity for visualization of specific tissues, assessing a disease and/or monitoring effects of therapeutic treatments, or high radiation dose, for delivering adequate doses of ionizing radiation to a specified diseased site, without the risk of radiation injury/radiotoxicity in other e.g. healthy tissues. It is thus of crucial interest to determine and assess cell-specific structures and in particular structures that are present in case of cancer (i.e. tumors) or inflammatory and autoimmune diseases, such as receptors, antigens, haptens and the like which can be specifically targeted by the respective biological vehicles.
The folate receptor (FR) has been identified as one of these structures (Low, Acc Chem Res. 2008; 41:120-9). The FR is a high-affinity (KD<10−9 M) membrane-associated protein. In normal tissues and organs FR-expression is highly restricted to only a few organs (e.g. kidney, lungs, choroids plexus, and placenta), where it largely occurs at the luminal surface of epithelial cells and is therefore not accessible for folate in the circulation. The FR-alpha is frequently overexpressed on a wide variety of specific cell types, such as epithelial tumors (e.g. ovarian, cervical, endometrial, breast, colorectal, kidney, lung, see e.g. Parker et al., Anal. Biochem. 2005; 2:284-293), whereas the FR-beta is frequently overexpressed in leukaemia cells (approx. 70% of acute myelogenous leukaemia (AML) are FR-beta positive). Both may therefore be used as a valuable tumor marker for selective tumor-targeting (Elnakat and Ratnam, Adv. Drug Deliv. Rev. 2004; 56:1067-84). In addition it has recently been discovered that activated (but not resting) synovial macrophages in patients diagnosed with rheumatoid arthritis possess a functionally active FR-beta (Nakashima-Matsushita et al, Arthritis & Rheumatism, 1999, 42(8): 1609-16). Therefore activated macrophages can be selectively targeted with folate conjugates in arthritic joints, a capability that opens possibilities for the diagnosis and treatment of rheumatoid arthritis (Paulos et al, Adv. Drug Deliv. Rev. 2004; 56:1205-17). Other inflammatory pathologies in which folate receptor positive macrophages are commonly enriched include rheumatoid arthritis, Crohn's disease, atherosclerosis, sarcoidosis, glomerulonephritis, osteoarthritis, organ transplant rejection, ulcerative colitis, Sjogren's syndrome, diabetes, ischemia/reperfusion injury, impact trauma, microbial infection, prosthesis osteolysis, liver steatosis, and multiple sclerosis (Piscaer et al. 2011, Arthritis & Rheumatism 63, 1898; Henne et al. 2012, Mol Pharm, 9:1435-40; Ayala-Lopez et al. 2010, J Nucl Med 51, 768). Folate-targeted therapeutic agents offer great promise for the development of highly potent, nontoxic treatment modalities for the same diseases (Hansen M. J et al., Targeted Drug Strategies for Cancer and Inflammation, Springer Science+Business Media, 2011, 181-193). FR-beta is also overexpressed on tumor-associated macrophages (TAMs). TAMs show mostly pro-tumoral functions, promoting tumor cell survival, proliferation, and dissemination. Clinical studies have shown a correlation between the numbers of TAMs and poor prognosis for amongst others breast, prostate, ovarian, cervical, endometrial, esophageal, pancreatic, glioblastoma and bladder cancers (Kurahara H. et al., Ann Surg Oncol., 2012 Feb. 16, Nagai T. et al., Cancer Immunol Immunother (2009) 581577-1586, Puig-Kroeger A. et al. Cancer Res 2009; 69 (24). Dec. 15, 2009, Turk M. J. et al., Cancer Letters 213 (2004) 165-172). Therefore tumor-associated macrophages can be selectively targeted with folate conjugates. That opens possibilities for the diagnosis and treatment of cancer.
Another such cell-specific structure is the proton-coupled folate transporter (PCFT). PCFT is expressed in the proximal small intestine, where it mediates folate absorption at acidic pH (Qiu et al, Cell. 2006 Dec. 1; 127(5):917-28) and in tissues such as liver and kidney, which do not experience low pH conditions (Zhao et al., Expert Rev Mol Med. 2009 Jan. 28; 11:e4). The interstitial pH of solid tumors is often acidic (Helmlinger et al., Nat Med. 1997 February; 3(2):177-82; Raghunand et al., Biochem Pharmacol. 1999 Feb. 1; 57(3):309-12), which favors PCFT transport. A prominent low-pH transport route was identified in 29 of 32 solid human tumor cell lines (Zhao et al., Clin Cancer Res. 2004 Jan. 15; 10(2):718-27), and high levels of human PCFT (hPCFT) transcripts were reported in a broad range of human tumors (Kugel Desmoulin et al., Am Assoc Cancer Res 51:1103). The role of hPCFT in antifolate activity and tumor selectivity is still evolving. Transport of classic antifolates by PCFT has been described previously (Zhao et al., Mol Pharmacol. 2008 September; 74(3):854-62. Epub 2008 Jun. 4). A targeting agent of the proton-coupled folate transporter opens possibilities for the diagnosis and treatment of tumors.
Folates and its derivatives have thus been intensively studied over the past 15 years as targeting agents for the delivery of therapeutic and/or diagnostic agents to cell populations bearing folate receptors in order to achieve a selective accumulation of therapeutic and/or diagnostic agents in such cells relative to normal cells.
Various probes have been conjugated to folic acid and (pre)clinically evaluated, including folate radiopharmaceuticals (Leamon and Low, Drug Discov. Today 2001; 6:44-51 and Jammaz et al, J. Label Compd. Radiopharm. 2006; 49:125-137; Müller & Schibli, 2011 J. Nucl. Med. 52, 1; Müller, Current Pharm Design, 2012), folate-conjugates of chemotherapeutic agents (Leamon and Reddy, Adv. Drug Deliv. Rev. 2004; 56:1127-41; Leamon et al, Bioconjugate Chem. 2005; 16:803-11), proteins and protein toxins (Ward et al, J. Drug Target. 2000; 8:119-23; Leamon et al, J. Biol. Chem. 1993; 268:24847-54; Leamon and Low, J. Drug Target. 1994; 2:101-12), antisense oliconucleotides (Li et al, Pharm. Res. 1998; 15:1540-45; Zhao and Lee, Adv. Drug Deliv. Rev. 2004; 56:1193-204), liposomes (Lee and Low, Biochim. Biophys. Acta-Biomembr. 1995; 1233:134-44; Gabizon et al, Adv. Drug Deliv. Rev. 2004; 56:1177-92), hapten molecules (Paulos et al, Adv. Drug Deliv. Rev. 2004; 56:1205-17), MRI contrast agents (Konda et al, Magn. Reson. Mat. Phys. Biol. Med. 2001; 12:104-13) etc.
Folate radiopharmaceuticals can be in particular very useful for an improved diagnosis and evaluation of the effectiveness of cancer therapy. This may include assessment and/or prediction of a treatment response and consequently improvement of radiation dosimetry. A typical visualization technique is positron emission tomography (PET), whereby a positron emitting radionuclide is administered to a subject, and as it undergoes radioactive decay the gamma rays resulting from the positron annihilation are detected in the PET scanner. Due to its high sensitivity and well-elaborated quantification methods, PET has established itself as one of the most sophisticated functional imaging technologies to assess regional uptake and affinity of ligands or metabolic substrates in brain and other organs and thus provides measures of imaging based on metabolic activity. Suitable radiopharmaceuticals for PET may be based on a metal isotope in combination with a chelator for entrapment of the metal (e.g. 68Ga, 64Cu, 89Zr), or on a covalently linked isotope, typically positron emitting isotopes with short half lives such as 11C (ca. 20 min), 13N (ca. 10 min), 15O (ca. 2 min) and 18F (ca. 110 min).
Over the past decades, a number of chelate-based folate radiopharmaceuticals, in particular 111In-, 99mTc- and 67/8Ga8Ga-derivatives, have been synthesized and successfully evaluated as diagnostic agents for imaging folate receptor-positive tumors using SPECT or PET (see e.g. Siegel et al., J. Nucl. Med. 2003, 44:700; Müller et al., J. Organomet. Chem. 2004, 689:4712; Mathias et al., Nucl. Med. Biol. 2003, 30(7):725; WO 2008/125618; Müller et al. 2011, Nucl. Med. & Biol. 38, 715).
More recently, folate radiopharmaceuticals carrying a covalently linked positron emitting 18F nuclide have been reported (see e.g. Bettio et al., J. Nucl. Med., 2006, 47(7), 1153; WO 2006/071754; WO 2008/098112; WO 2008/125613; WO 2008/125615; WO 2008/125617; WO 2010/040854; Ross et al. 2010, J Nucl. Med., 51, 1756; Fischer et al. 2012, Bioconjug. Chem.), and shown to be most suitable for PET imaging because of its excellent imaging characteristics which would fulfill all of the above mentioned considerations.
Yet, while known 18F folate radiopharmaceuticals show promising results, there is still a need for compounds that show high FR-specificity and are suitable for routine clinical applications and yet can be obtained in efficient and versatile ways with high radiochemical yields
Applicants have now found that folate derivatives wherein the phenyl group of the folate skeleton has been replaced by a heterocycle can be substituted with one or more 18F nuclides in versatile and efficient ways and high radiochemical yields. The obtained 18F-folate/antifolate analogue compounds show high selectivity for FR-positive tissue and thus it can be concluded such modifications to the folate skeleton exert no negative effect on folate receptor binding affinity.
Thus, the present invention is directed to new 18F-folate/antifolate analogue radiopharmaceuticals, wherein the phenyl group, which connects the condensed pyrimidine heterocycle via suitable linkers (such as a —CH2—NH-linker at the C6 position of a pteridine heterocycle) to the amino acid portion within folate structures, has been replaced by an 18F-substituted 5- or 6-membered heterocycle, their precursors, a method of their preparation, as well as their use in diagnosis of a cell or population of cells expressing a folate-receptor and monitoring of cancer and inflammatory and autoimmune diseases and therapy thereof.